PROJECT SUMMARY/ABSTRACT Nucleus pulposus (NP) cells reside in a soft, gelatinous matrix that dehydrates and becomes increasingly fibrotic with age. Age-related changes in these ?physical cues? of matrix stiffness may be potent regulators of NP cell phenotype, and may contribute to transitions toward a senescent and fibroblastic NP cell with a limited capacity for repair. This project proposed in the NRSA Fellowship will study how ?physical cues? of matrix stiffness can regulate NP cell phenotype and identify mechanisms that can preserve a biosynthetically active, juvenile NP cell phenotype. We have shown that NP cells forming multi-cell clusters on ?soft? laminin-presenting substrates demonstrate biosynthetically active, juvenile NP phenotype with cytosolic localization of myocardin related transcription factor (MRTF-A), a co-activator of serum response factor (SRF) that promotes fibroblast-like behaviors in many cells. Additionally, it has been shown that other nuclear factors and signaling pathways may mediate mechanoresponses to matrix physical properties. In Specific Aim 1, we will determine if MRTF-A co-activation of SRF is necessary for promoting the fibroblastic differentiation of human NP cells, measured as decreased expression of NP-specific markers and reduced ECM biosynthesis, when cultured upon ?stiff? substrates. Primary human NP cells will be transduced with SRE-luc or LifeAct GFP reporter plasmids, and then MRTF-A will be knocked down with CRISPRi/Cas9. Cells will be cultured on ?stiff? PEG-laminin hydrogels and assessed for key NP-specific matrix synthesis and molecular markers, transcription factor and signaling molecule activity, and cytoskeletal organization. RNA-seq analysis will determine any downstream pathways implicated in MRTF-SRF signaling. In Specific Aim 2, we will evaluate if blocking YAP/TAZ activation of TEAD can promote NP-specific marker expression and elevated biosynthesis for adult human NP cells cultured on ?stiff? substrates. Human NP cells will carry either TEAD-luc or LifeAct-GFP reporter plasmids and then transduced with lentivirus carrying YAP CRISPRi/Cas9 plasmid to disrupt YAP/TAZ signaling. These cells will be cultured on ?stiff? laminin-presenting hydrogels (as well as ?soft? for a control) and measured for the same NP-specific markers and RNA-seq analysis as in Specific Aim 1. The applicant will work with the Sponsor and mentoring team to learn new techniques in state-of-the-art genome engineering and RNA-seq methods as well as methods to quantify cytoskeletal organization that will advance both the applicant's career and the translation of cell phenotype manipulation for application to disc regeneration. The applicant will obtain broader training in research methodology, grant and manuscript preparation and responsible conduct in research that will prepare her for a career as an independent researcher.